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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Reductionist Approach in Peptide-Based Nanotechnology

Gazit, Ehud
Annu. Rev. Biochem..
Jun 2018
10.1146/annurev-biochem-062917-012541
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like β-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.

The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity: Bae et al.

Bae, Heekyong R.; Hodge, Deborah L.; Yang, Guo-Xiang; Leung, Patrick S.C.; Chodisetti, Sathi Babu; Valencia, Julio C.; Sanford, Michael; Fenimore, John M.; Rahman, Ziaur S.M.; Tsuneyama, Koichi; Norman, Gary L.; Gershwin, M. Eric; Young, Howard A.
Hepatology.
Apr 2018
10.1002/hep.29524
We have reported on a murine model of autoimmune cholangitis, generated by altering the AU-rich element (ARE) by deletion of the interferon gamma (IFN-γ) 3′ untranslated region (coined ARE-Del−/−), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the sex bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del−/− mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del−/− Ifnar1−/− mice dramatically reduces liver pathology and abrogated sex bias. More importantly, female ARE-Del−/− mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del−/− Ifnar1−/− mice corrects these GC abnormalities, including abnormal follicular structure. Conclusion: Our data implicate type I IFN signaling as a necessary component of the sex bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. (Hepatology 2018;67:1408-1419)

Bicaudal D2 is a novel autoantibody target in systemic sclerosis that shares a key epitope with CENP-A but has a distinct clinical phenotype

Fritzler, Marvin J.; Hudson, Marie; Choi, May Y.; Mahler, Michael; Wang, Mianbo; Bentow, Chelsea; Milo, Jay; Baron, Murray; Pope, J.; Baron, M.; Markland, J.; Robinson, D.; Jones, N.; Khalidi, N.; Docherty, P.; Kaminska, E.; Masetto, A.; Sutton, E.; Mathieu, J.-P.; Hudson, M.; Ligier, S.; Grodzicky, T.; LeClercq, S.; Thorne, C.; Gyger, G.; Smith, D.; Fortin, P.R.; Larché, M.; Abu-Hakima, M.; Rodriguez-Reyna, T.S.; Cabral, A.R.; Fritzler, M.J.
Autoimmunity Reviews.
Mar 2018
10.1016/j.autrev.2018.01.006
We studied the clinical correlations and epitopes of autoantibodies directed to a novel autoantigen, Bicaudal D (BICD2), in systemic sclerosis (SSc) and reviewed its relationship to centromere protein A (CENP-A). 451 SSc sera were tested for anti-BICD2 using a paramagnetic bead immunoassay and then univariate and multivariate logistic regression was used to study the association between anti-BICD2 and demographic and clinical parameters as well as other SSc-related autoantibodies. Epitope mapping was performed on solid phase matrices. 25.7% (116/451) SSc sera were anti-BICD2 positive, of which 19.0% had single specificity anti-BICD2 and 81.0% had other autoantibodies, notably anti-CENP (83/94; 88.3%). Compared to anti-BICD2 negative subjects (335/451), single specificity anti-BICD2 subjects were more likely to have an inflammatory myopathy (IM; 31.8% vs. 9.6%, p = .004) and interstitial lung disease (ILD; 52.4% vs. 29.0%, p = .024). Epitope mapping revealed a serine- and proline-rich nonapeptide SPSPGSSLP comprising amino acids 606–614 of BICD2, shared with CENP-A but not CENP-B. We observed that autoantibodies to BICD2 represent a new biomarker as they were detected in patients without other SSc-specific autoantibodies and were the second most common autoantibody identified in this SSc cohort. Our data indicate that the major cross-reactive epitope is associated with anti-CENP-A but, unlike anti-CENP, single specificity anti-BICD2 antibodies associate with ILD and IM.

Identification of the antigenic epitopes of maternal autoantibodies in autism spectrum disorders

Edmiston, Elizabeth; Jones, Karen L.; Vu, Tam; Ashwood, Paul; Van de Water, Judy
Brain, Behavior, and Immunity.
Mar 2018
10.1016/j.bbi.2017.12.014
Several groups have described the presence of fetal brain-reactive maternal autoantibodies in the plasma of some mothers whose children have autism spectrum disorder (ASD). We previously identified seven autoantigens targeted by these maternal autoantibodies, each of which is expressed at significant levels in the developing brain and has demonstrated roles in typical neurodevelopment. To further understand the binding repertoire of the maternal autoantibodies, as well as the presence of any meaningful differences with respect to the recognition and binding of these ASD-specific autoantibodies to each of these neuronal autoantigens, we utilized overlapping peptide microarrays incubated with maternal plasma samples obtained from the Childhood Autism Risk from Genetics and Environment (CHARGE) Study. In an effort to identify the most commonly recognized (immunodominant) epitope sequences targeted by maternal autoantibodies for each of the seven ASD-specific autoantigens, arrays were screened with plasma from mothers with children across diagnostic groups (ASD and typically developing (TD)) that were positive for at least one antigen by western blot (N = 67) or negative control mothers unreactive to any of the autoantigens (N = 18). Of the 63 peptides identified with the discovery microarrays, at least one immunodominant peptide was successfully identified for each of the seven antigenic proteins using subsequent selective screening microarrays. Furthermore, while limited by our relatively small sample size, there were peptides that were distinctly recognized by autoantibodies relative to diagnosis For example, reactivity was observed exclusively in mothers of children of ASD towards several peptides, including the LDH-B peptides DCIIIVVSNPVDILT (9.1% ASD vs. 0% TD; odds ratio (95% CI) = 6.644 (0.355–124.384)) and PVAEEEATVPNNKIT (5.5% ASD vs. 0% TD; odds ratio (95% CI) = 4.067 (0.203–81.403)).These results suggest that there are differences in the binding repertoire between the antigen positive ASD and TD maternal samples. Further, the autoantibodies in plasma from mothers of children with ASD bound to a more diverse set of peptides, and there were specific peptide binding combinations observed only in this group. Future studies are underway to determine the critical amino acids necessary for autoantibody binding, which will be essential in developing a potential therapeutic strategy for maternal autoantibody related (MAR) ASD.

A Trifunctional Linker for Purified 3D Assembled Peptide Structure Arrays

Mattes, Daniela S.; Rentschler, Simone; Foertsch, Tobias C.; Münch, Stephan W.; Loeffler, Felix F.; Nesterov-Mueller, Alexander; Bräse, Stefan; Breitling, Frank
Small Methods.
Feb 2018
10.1002/smtd.201700205
Microarrays are an important tool in modern research that allow the rapid screening of many different interactions simultaneously. Peptide arrays, which bear different peptides arranged in separate spots, permit high-throughput screening to investigate linear and cyclic binding sites. To study conformational or discontinuous binding sites, protein arrays are the major choice. However, the tremendous costs for the generation of high-density protein arrays of high purity restrict progress in protein research. Therefore, peptide-based arrays, which can mimic assembled peptide structures, have an enormous potential. Here, a method is presented to create such structures in the array format as an alternative to protein arrays. A trifunctional linker is developed with an azide, a protected alkyne, and a carboxyl group, which can react with two or three different peptides. Due to the spatial proximity, the peptides interact and can form an assembled peptide structure. As a proof of concept, assembled peptide structures are demonstrated on beads and on a polymer surface and the approach can be validated via matrix-assisted laser desorption/ionization spectrometry. Furthermore, a multistep transfer of peptide arrays is shown, generating purified assembled peptide structure arrays in high density.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
10.1038/s41598-017-17071-0
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Weber, Laura K.; Isse, Awale; Rentschler, Simone; Kneusel, Richard E.; Palermo, Andrea; Hubbuch, Jürgen; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Eng. Life Sci..
Oct 2017
10.1002/elsc.201700062
Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

Peptide array functionalization via the Ugi four-component reaction

Ridder, B.; Mattes, D. S.; Nesterov-Mueller, A.; Breitling, F.; Meier, M. A. R.
Chem. Commun..
May 2017
10.1039/C7CC01945A
The Ugi four-component reaction was investigated as a tool for the functionalization of peptide arrays via post-synthetic side-chain modification, mimicking post-translational processes. Additionally, as a proof of concept for the synthesis of peptidomimetics on arrays, the integration of an Ugi unit into a growing peptide chain was demonstrated.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays

Weber, Laura K.; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen; Dübel, Stefan; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Journal of Immunological Methods.
Apr 2017
10.1016/j.jim.2017.01.012
The antibody species that patrol in a patient’s blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding “their” antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis

Qendro, Veneta; Bugos, Grace A.; Lundgren, Debbie H.; Glynn, John; Han, May H.; Han, David K.
Proteomics.
Mar 2017
10.1002/pmic.201600322
In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases.

Acquired Factor XIII inhibitor associated with mantle cell lymphoma: ACQUIRED FXIII INHIBITOR

Nixon, Christian P.; Prsic, Elizabeth H.; Guertin, Christine A.; Stevenson, Ryan L.; Sweeney, Joseph D.
Transfusion.
Mar 2017
10.1111/trf.13947
BACKGROUND Acquired Factor (F)XIII deficiency is a very rare bleeding diathesis with a potentially fatal outcome, previously described in the context of autoimmune disorders and leukemias. There is minimal information on autoantibody characterization and the role of antifibrinolytic therapy in patient management. CASE REPORT A 79-year-old woman with a 3-month history of bruising and heavy menorrhagia presented with ongoing vaginal bleeding, symptomatic anemia, and a right thigh hematoma. Initial management included an axillary lymph node biopsy and coagulation evaluation. Pathologic examination of the biopsy specimen revealed mantle cell lymphoma. Clot solubility assay was consistent with a FXIII activity of less than 3%. An anti-FXIII inhibitor was suspected, the epitope specificity of which was mapped by micropeptide array analysis to regions in the β-sandwich and catalytic core domain of the FXIII-A subunit. Management with cryoprecipitate, steroids, rituximab, and antifibrinolytic therapy resolved the bleeding diathesis and suppressed the inhibitor. CONCLUSION This is the first reported case of an acquired FXIII inhibitor associated with mantle cell lymphoma in which the epitope specificity of the pathologic autoantibody was accurately defined. Antifibrinolytic therapy played a prominent role in the prevention of bleeding complications in the window period between initiation of immunosuppression and disappearance of the pathologic anti-FXIII autoantibody.

Autoantikehad täppismeditsiinis

Jaks, Viljar; Uibo, Raivo
Feb 2017
10.15157/EA.V0I0.13344
Immuuntolerantsi häirumine, mille üheks väljundiks on antikehade teke organismile omaste biomolekulide vastu, on oluline patogeneetiline mehhanism mitmete laialdaselt levinud haiguste puhul ja seetõttu on autoantikehade määramine kujunenud oluliseks diagnostiliseks vahendiks. Artiklis on käsitletud autoantikehade esinemise olulisust haiguste tekke ja kulu prognoosimisel. Kuigi sellekohane info on veel üsna napp, on selge, et organismi immuunstaatuse muutus eelneb aastaid haiguse ilmnemisele ning autoimmuunset komponenti sisaldava haiguse kulg ja prognoos on seotud patsiendil esinevate kindlate autoantikehadega. Sellest tulenevalt võime loota, et organismi immuunstaatuse uurimine, eriti aga autoantikehade spektri iseloomustamine, on tulevikus geneetilise info analüüsimise kõrval üks täppismeditsiini olulisemaid tööriistu.

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